Effects of acidification on nitrification and associated nitrous oxide emission in estuarine and coastal waters

In the context of an increasing atmospheric carbon dioxide (CO2) level, acidification of estuarine and coastal waters is greatly exacerbated by land-derived nutrient inputs, coastal upwelling, and complex biogeochemical processes. A deeper understanding of how nitrifiers respond to intensifying acidification is thus crucial to predict the response of estuarine and coastal ecosystems and their contribution to global climate change. Here, we show that acidification can significantly decrease nitrification rate but stimulate generation of byproduct nitrous oxide (N2O) in estuarine and coastal waters. By varying CO2 concentration and pH independently, an expected beneficial effect of elevated CO2 on activity of nitrifiers (“CO2-fertilization” effect) is excluded under acidification. Metatranscriptome data further demonstrate that nitrifiers could significantly up-regulate gene expressions associated with intracellular pH homeostasis to cope with acidification stress. This study highlights the molecular underpinnings of acidification effects on nitrification and associated greenhouse gas N2O emission, and helps predict the response and evolution of estuarine and coastal ecosystems under climate change and human activities.


Reviewer #1 (Remarks to the Author):
The topic of the manuscript "Effects of acidification on nitrification and associated 2 nitrous oxide emission in estuarine and coastal waters" by Zhou et al. is very interesting and worth study. Lots of tests were conducted and experimental data were obtained. The authors carried out analysis and mining of these experimental data and then gotten interesting results. However, theoretical description is not enough and the evidence is also insufficient. So, I think, the manuscript can only be accepted after some major revisions. 1] As described as "the measured inhibition of nitrification rate by acidification in the estuarine and coastal waters was generally lower than that in the oligotrophic open oceans where nitrification rates were reported to decline by 3-44% in response to a decrease of 0.1 pH unit", was here the differences in the natures of Nutrients among the water samples collected " 6 representative sites (Yz1 to Yz6)" ? Did these natures have influences on the nitrification rate To answer these issues, therefore, more composites of the samples should be given in the paper. Especially, "oligotrophic open oceans", "toxicant exposures", and "human activity-induced eutrophication" were employed to elucidate the influences of acidification on nitrification and its effects. 2] it is good that "nitrifying communities" " different nitrifying organisms" and even "heterotrophic denitrifiers" were employed to explain the pathway of N2O production. Even so, more evidences and data were wanted to prove that "the contribution of denitrifying bacteria to the production of N2O should be negligible." 3] to research "Decoupling potential effects of elevated pCO2 and reduced pH", the methods including adjusting pH (7.8 and 8.1) with sterile acid or base solution were used as Breider et al.37 did. In the present study, what had been done to avoid changing alkalinity and carbonate parameters of the studied water ? 4] "previous attempts to acquire metatranscriptomes based on the short-term acidification experiments failed, because there was no enough qualified mRNA with good integrity and purity, which might be due to the extreme instability of mRNA and the complexity of environmental samples18" . I think, these are just appearances, but the essence is as you described that " nitrifiers account for only a small fraction of the complex microbial communities in estuarine and coastal waters ". So, it is good that " a series of continuous-flow environmental simulators with water samples from site Yz3 were set up to mimic long-term acidification and to enrich nitrifiers." 5] when these experimental data Data analysis and mining were done, more comparsion studies can be done with the results of other researchers. 6] Line 267, what are short for "amoB (also suggested as a catalytic subunit47 267 ) and amoc" ? 7] Line 403, should " word" be replaced by "world"?
Reviewer #2 (Remarks to the Author): "Effects of acidification on nitrification and associated nitrous oxide emission in estuarine and coastal waters" By Zhou et al This manuscript reports on an intensive experimental investigation into the effect of ocean acidification on nitrification rates and nitrous oxide production in estuarine and coastal waters. A particular and noteworthy factor of this work relative to previous investigations is the detailed analysis of the metatranscriptomics associated with these processes. Whilst this manuscript is thorough in detail and the results are novel and of importance to our understanding of contemporary and future nitrogen biogeochemistry I find that the current presentation is far too reliant on supplementary material, requiring the reader to constantly change between the two documents. This current organisation renders this paper hard to follow and difficult to interpret. I did not even realise that there had been measurements of nitrification and nitrous oxide in an enriched culture additional to the unenriched experiment until reaching the end of my review, and whilst this enrichment experiment supports the overall findings there is no discussion about the relevance or potential experimental artefact offered by this treatment. My overriding suggestion is not to remove the supplementary material, but to integrate it better into the main paper and to re-organise so that the whole document is easier for the reader to follow.
Whilst there is a fairly comprehensive review and integration of previous literature, I would suggest that the work of Fulweiler et al (2011( -DOI 10.1007 and Rees et al (2016Rees et al ( -http://dx.doi.org/10.1016Rees et al ( /j.dsr2.2015.006) should both be incorporated into discussions here. Fulweiler's work showed that for estuarine/coastal conditions there was an inverse relationship between pH and nitrification, that is the opposite effect to that shown here. Whilst Rees showed that N2O concentration decreased with increasing OA and saw no change in numbers of AOA both of which are different to here. The study of Rees was in open water and in very different conditions to this paper, but as one of very few papers on this subject probably offers some discussion points pertinent to the control of N2O release from nitrification.
Specific comments: L33 Whilst offering important insights the claim that this work will "help predict climate change" is taking things too far. L121-122 eg Fulweiler et al L129 disturbance on nitrification COULD cause profound consequences L133 Beman predicted, Rees et al confirmed for open ocean conditions L234-235 …. There was NOT enough mRNA with good integrity …. L245 I'm not convinced that the data shown in Supplementary Fig 8 shows that steady state was reached after 25 days. There needs to be some informed discussion on this figure. I can't see why ammonium concentration in "Influent" was as high as 60µM, the ambient at Yz3 was ~10µM, but then where does it go, as is close to zero in effluentsurely not all is nitrified?? L218-224. This is hard to follow and needs re-writing L226 Transcriptional response of nitrifiers to acidification during long-term incubation L278 Supplementary Fig 15 referred to before 12, 13, 14 L564 nutrient analysis likely to be colorimetrically rather than spectrophotometrically  Table 9, 10, 11 -no reference to this table found Reviewer #3 (Remarks to the Author): This paper addresses an important issue relating to climate change and environmental impacts in the coastal zone. Authors used a suite of technologies to investigate nitrification and emission of nitrous oxides under ocean acidification, overall the paper has potential in getting published in Nature Communications, but have the following concerns.
Major comments: 1. Although the authors distinguished the effects of acidification from pCO2 and pH only, acidification decreased nitrification rate and increased N2O production. This main result has been shown in a previous study (https://doi.org/10.1038/s41558-019-0605-7).
2. I was confused by the sampling strategy. It seems that the bottom (sedimentary) water was taken? why not sample the surface water that is mainly affected by acidification. Also, it is widely accepted that nitrification functions well in the surface, and it is less likely in the bottom water.
3. Why not consider Comammox process, not only on transcript level? I think this is important when evaluating nitrification process. Did authors conduct amplicon sequencing?
Minor comments: 1. what does "CO2-fertilization" mean This is mainly used to describe this effect in terrestrial ecosystem. It should be avoided using any ambiguous words in abstract; 2. what does "efficient changes" in gene expression refer to in the abstract This sentence here does not mean much sense, it does not convey any practical info; 3. Misleading statement. "However, how microbially-mediated biogeochemical processes may be altered by acidification in estuarine and coastal ecosystems remains largely unknown, to a great extent limiting our understanding of the responses of these ecosystems to global environmental changes". Many studies have explored the microbially-mediated biogeochemical processes in estuarine and coastal ecosystems. E.g., DOI10.1038/s41561-020-0584-3; DOI10.1021/acs.est.2c00692; DOI10.1002/2014GL060849; DOI10.1016/j.scitotenv.2020.142689; DOI10.1038/s41467-017-00417-7 4. Line 95, delete "new"; 5. Line 377 Indeed, the comammox amoA gene abundance was higher in the acidified treatments, how did the authors confirm that this amoA gene is affiliated to comammox, not for AOA or AOB?
Overall, I think is work is interesting and authors should improve the MS to distill the major significance better, as opposed to previously reported results, as indicated earlier.

Point-by-point response to the reviewers' comments Reviewer #1 (Remarks to the Author):
Comment: The topic of the manuscript "Effects of acidification on nitrification and associated nitrous oxide emission in estuarine and coastal waters" by Zhou et al. is very interesting and worth study. Lots of tests were conducted and experimental data were obtained. The authors carried out analysis and mining of these experimental data and then gotten interesting results. However, theoretical description is not enough and the evidence is also insufficient. So, I think, the manuscript can only be accepted after some major revisions.
Response: Thank you very much for your comments. This manuscript has been carefully modified according to your suggestions, and all the raised issues have been addressed as follows.

Comment: 1] As described as "the measured inhibition of nitrification rate by acidification in the estuarine and coastal waters was generally lower than that in the oligotrophic open oceans where nitrification rates were reported to decline by 3-44%
in response to a decrease of 0.1 pH unit", was here the differences in the natures of Nutrients among the water samples collected "6 representative sites (Yz1 to Yz6)"? Did these natures have influences on the nitrification rate To answer these issues, therefore, more composites of the samples should be given in the paper. Especially, "oligotrophic open oceans", "toxicant exposures", and "human activity-induced eutrophication" were employed to elucidate the influences of acidification on nitrification and its effects.

Response:
In this study, we determined the concentrations of nitrogen nutrients in all water samples collected from sites Yz1 to Yz6, which showed a decreasing trend from the upper estuary to the coastal area, due to the watershed anthropogenic nutrient inputs.
This difference leads to higher nitrification rate in the upper estuary waters (please see Supplementary Fig. 2) and also affects the response of nitrifiers to acidification along the nutrient gradient. In the revised manuscript, the correlation between NH4 + concentration and the effects of acidification on nitrification rates has been supplemented based on this study and the previously reported data. Results showed a significant negative relationship between NH4 + concentration and the inhibition effect of acidification on nitrification rate (please see Supplementary Fig. 4). In addition, environmental parameters of the water samples are provided in Supplementary Table 1, and the following information has also been provided in the revised manuscript:  Fig. 4)" (Please see lines 124-132).
Comment: 2] it is good that "nitrifying communities" "different nitrifying organisms" and even "heterotrophic denitrifiers" were employed to explain the pathway of N2O production. Even so, more evidences and data were wanted to prove that "the contribution of denitrifying bacteria to the production of N2O should be negligible."

Response:
To explore the pathway of N2O production, 15 N-site preference (SP) value was calculated by the isotopomer ratios as shown below: for NH2OH oxidation and 0‰ for NO2 P reduction, which were estimated in pure cultures (Sutka et al., 2006;Rathnayake et al., 2013), were used to estimate the contribution of each process, assuming that each process is linearly proportional to the SP value using the following equations (Rathnayake et al., 2013).
On this basis, the calculated SP values in our continuous-flow manipulation systems were from 28.6 to 33.8. Thus, it was estimated that 86.7-100% of the released N2O was produced via NH2OH oxidation, whereas the contribution of NO2 P reduction was minor or nil. It should be noted that, the N2O isotopomer analysis could not distinguish the relative contributions of nitrifier denitrification and heterotrophic denitrification.
Although heterotrophic denitrifiers may also contribute to the production of N2O, their contribution may be insignificant as the samples from all study sites were well oxygenated (DO: 8.30-9.86 mg L -1 ) and remained at near in situ high DO levels during the incubation. Actually, previous studies reported that when the dissolved O2 concentration is more than 0.06 mg L -1 , the N2O production by denitrification is completely inhibited (Dalsgaard et al. 2014). Therefore, the contribution of heterotrophic denitrifying bacteria to the production of N2O should be negligible. This point has been clarified in the revised manuscript: "Although heterotrophic denitrifiers may also contribute to the production of N2O, their contribution may be insignificant, as the natural isotopic signatures of N2O showed that the pathway of NO2 5 reduction (including nitrifier denitrification and heterotrophic denitrification) contributed only 0-13.3% of the released N2O (Supplementary Table 4 Previous studies reported that when the DO concentration is more than 0.06 mg L -1 , the N2O production by heterotrophic denitrification is completely inhibited 49 . Therefore, the contribution of denitrifying bacteria to the production of N2O should be negligible"  (2011( -DOI 10.1007 and Rees et al (2016Rees et al ( -http://dx.doi.org/10.1016Rees et al ( /j.dsr2.2015 should both be incorporated into discussions here. Fulweiler's work showed that for estuarine/coastal conditions there was an inverse relationship between pH and nitrification, that is the opposite effect to that shown here. Whilst Rees showed that N2O concentration decreased with increasing OA and saw no change in numbers of AOA both of which are different to here. The study of Rees was in open water and in very different conditions to this paper, but as one of very few papers on this subject probably offers some discussion points pertinent to the control of N 2 O release from nitrification. Response: Thank you very much for this suggestion. In the revised manuscript, these recommended studies have all been incorporated into the discussions: "Whilst nitrification rate was reported to increase along a decreasing natural gradient of pH in Narragansett Bay 43 , it was likely due to a combination of biogeochemical conditions rather than the effect of acidification alone" (Please see lines 116-118); "However, Rees et al. 44 documented inhibition of N2O production by ocean acidification in cold temperate and polar seawaters. Assuming that nitrification is the main N2O production pathway in their study, the response of the N2O production to acidification would be different in polar seas" (Please see lines 171-174). In addition, Rees's work showed that AOA assemblage composition lacked sensitivity to short-term ocean acidification, and this point has also been incorporated into the discussions: "However, Rees et al. 44 reported that AOA assemblage composition was not sensitive to ocean acidification, possibly because the incubation period (less than one week) in their study was not long enough to cause significant turnover in the assemblage" (Please see lines 387-390).
(2) Compared with open ocean, the environmental conditions as well as the nitrifier communities are quite different in the complex estuarine and coastal waters. For example, the ammonium concentration could be several orders of magnitude higher in the estuarine and coastal regions due to intensive human activities, and "Moreover, nitrifying communities in diverse aquatic habitats may respond differently to acidification, as the mechanisms for N2O production differ among different nitrifying organisms 32,38,40 . Therefore, the response of N2O production during nitrification to aquatic acidification in estuarine and coastal waters needs to be evaluated" (Please see Response: Thank you very much for this comment. Unlike ocean acidification which is mainly driven by elevated atmospheric CO2 concentrations, estuarine and coastal water is also significantly affected by land-derived inputs and complex biogeochemical and hydrological processes (Scanes et al., 2020). One of the greatest threats to estuarine and coastal ecosystems worldwide is the excess nitrogen loading and consequent eutrophication (Al-Haj & Fulweiler, 2020). The eutrophication-induced phytoplankton production can result in high respiration rate and strong CO2 production in bottom waters (Laurent et al., 2017). Thus, near-bottom water generally suffers from more severe acidification in estuarine and coastal regions (Guo et al., 2021).
In addition, it was previously reported that nitrification is in general more active near the bottom of the shelf region (<60 m) (Shiozaki et al., 2019) or near the bottom of the euphotic zone of the open oceans (approximately 50~250 m) (Beman et al., 2011;Rees et al., 2016;Breider et al., 2019). Similarly, in the Yangtze Estuary and adjacent coastal waters, higher nitrification rates were generally observed in the bottom water (Wang et al., 2018). Therefore, near-bottom water samples (water depth: 9~31 m) were collected in the present study. This point has been clarified in the revised manuscript: "In this study, near-bottom waters, which typically exhibit more active nitrification and more severe acidification 65,66 , were collected from these sites…" (Please see lines 433-434).

Comment: 3. Why not consider Comammox process, not only on transcript level? I think this is important when evaluating nitrification process. Did authors conduct amplicon sequencing?
Response: Thank you very much for this comment. Actually, the newly discovered complete ammonia oxidizers (comammox) were considered in the present study. The abundance of comammox bacteria, as well as the canonical ammonia oxidizers: ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were quantified via qPCR. Results showed that the communities of the ammonia oxidizers changed significantly from the estuary mouth to its adjacent coastal area ( Supplementary Fig. 3). AOB were the dominant ammonia oxidizers in the upper estuary waters whereas the adjacent coastal regions were dominated by AOA. However, the abundance of comammox bacteria was low in the estuarine and coastal waters, which were only detected at the upper estuarine sites (Yz1-Yz3) but not in the adjacent coastal waters (Yz4-Yz6) ( Supplementary Fig. 3), suggesting that comammox bacteria may play an insignificant role in estuarine and coastal waters. In the present study, based on environmental water samples containing in situ nitrifying communities (including comammox bacteria), the responses of nitrification rate and associated N2O production to acidification were investigated.
In addition, amplicon sequencing was conducted to explore the whole nitrifying communities, not just comammox bacteria, using the universal primers capable of detecting both bacteria and archaea within the same sequencing libraries. Furthermore, the transcriptional responses of the key nitrifying genes (amo, hao, nxr) of comammox bacteria to acidification were mined from the metatranscriptome data: "In addition, based on metatranscriptome data, higher comammox gene transcripts were detected in the acidified treatments (0.78-11.59% of the total obtained gene transcripts involved in the stepwise oxidation of NH3 to NO3 5 ) compared with the ambient control (only 0.03-0.89%, P<0.05) (Fig. 6b and Supplementary Fig. 17)" (Please see lines 398-402).

Minor comments:
Comment: 1. what does "CO2-fertilization" mean This is mainly used to describe this effect in terrestrial ecosystem. It should be avoided using any ambiguous words in abstract; Response: The explanation of "CO2-fertilization" has been provided in the revised manuscript: "Higher pCO2 condition is expected to benefit nitrification, as an increased carbon source may promote the growth of chemoautotrophic nitrifiers (CO2fertilization) 24-27 " (Please see lines 67-69). In many previous studies, "CO2-fertilization" has been used to describe the effect of increased CO2 on chemoautotrophs in aquatic habitats (e.g., Hutchins et al., 2009, Beman et al., 2011Rees et al., 2016;Breider et al., 2019). To avoid ambiguity in the abstract, an explanation has also been provided as suggested: "beneficial effect of elevated CO2 on activity of nitrifiers ("CO2-fertilization" effect)" (Please see lines 30-31).

Comment: 2. what does "efficient changes" in gene expression refer to in the abstract
This sentence here does not mean much sense, it does not convey any practical info; Response: Thank you very much for this comment. As suggested, "make efficient changes in gene expressions to cope with acidification stress" has been changed to "nitrifiers could significantly up-regulate gene expressions associated with intracellular pH homeostasis to cope with acidification stress" (Please see lines 32-33).
Comment: 3. Misleading statement. "However, how microbially-mediated biogeochemical processes may be altered by acidification in estuarine and coastal ecosystems remains largely unknown, to a great extent limiting our understanding of the responses of these ecosystems to global environmental changes". Many studies have explored the microbially-mediated biogeochemical processes in estuarine and coastal ecosystems. E.g., DOI10.1038/s41561-020-0584-3;DOI10.1021/acs.est.2c00692;DOI10.1002DOI10.1016DOI10. /j.scitotenv.2020DOI10.1038/s41467-017-00417-7 Response: As suggested, this statement has been modified, and the recommended researches have been included in the revised manuscript: "Acidification in estuarine and coastal waters can thus be greatly intensified by episodic intrusion of high-CO2 upwelled water 11,13,14 , which may detrimentally affect biological processes and functioning of estuarine and coastal ecosystems 15-21 " (Please see lines 56-59).

Comment: 4. Line 95, delete "new";
Response: It has been removed from the revised manuscript, as suggested.
Comment: 5. Line 377 Indeed, the comammox amoA gene abundance was higher in the acidified treatments, how did the authors confirm that this amoA gene is affiliated to comammox, not for AOA or AOB?
Response: Copy numbers of comammox amoA gene were quantified by qPCR using the specific primer set A378F/C616R designed for comammox bacteria, and the specificity of this primer set was confirmed using clone libraries (Xia et al., 2018). In addition, taxonomic affiliations to the amoA gene transcripts were assigned by binning to the best hit in the NR database (BLASTP, e%H6?G: L )( -5 ) and were further confirmed by the phylogenetic analyses. Based on the constructed maximum-likelihood phylogenetic tree of amoA gene, the gene sequences affiliated to comammox bacteria can be clearly identified. In the revised manuscript, the phylogenetic tree of amoA gene has been provided as Supplementary Fig. 17.

Comment: Overall, I think work is interesting and authors should improve the MS to
distill the major significance better, as opposed to previously reported results, as indicated earlier.
Response: Thank you very much for your comments. As shown above, the manuscript has been greatly improved to better distill the major significance according to all these comments and suggestions.